The mosquito Aedes aegypti is the primary vector of human viruses causing dengue and yellow fever, diseases of global public health importance. Aedes aegypti is a peri-domestic species that lays desiccation- resistant eggs in water-filled human-made containers. In the proposed research, we will conduct a comprehensive investigation of the interaction of bacteria and Ae. aegypti eggs. The main goal of the proposed project is to identify two groups of bacteria-produced chemical cues, one that stimulates and another that inhibits egg hatching. Our hypothesis, based on extensive preliminary results, that bacteria-associated chemical cues regulate hatching is a departure from the generally accepted paradigm that egg hatching is stimulated by low dissolved oxygen (DO) concentration resulting from rapid bacterial growth. Our preliminary data show that a 1-day old mixed culture of 14 bacterial species, originally isolated from an experimental mosquito habitat, stimulates egg hatching under both high and low DO conditions. However, under supposedly stimulatory low DO conditions, eggs fail to hatch when submerged in an 8-day old culture of the same mix of bacterial species, suggesting that the same bacteria species can produce stimulatory and inhibitory cues, depending on the age of culture. In the proposed research, we will screen 14 bacterial species isolates from experimental mosquito habitats for bioactivity in stimulating and inhibiting egg hatch. Bacterial species exhibiting highly stimulatory or inhibitory bioactivity will be selected for further research. Chemical compounds associated with bacterial species will be identified by using bioassay-guided solvent extraction and fractionation coupled with chemical analytical techniques, including high-performance liquid chromatography and gas chromatography, and mass spectrometry. The activity of putative bioactive compounds will be verified in egg hatching bioassays. Once identified, the cue(s) that control hatching may be used to disrupt hatching of the pharate larva from the mosquito egg and offer innovative tools for control of a virus vector of global importance. The proposed research will provide a more complete understanding of the strategies that Ae. aegypti eggs employ to maximize survival under changing environmental conditions. Moreover, the project will provide tools and reagents for future research on the physiological events during the transition from embryo to neonate, including gene expression patterns and studies of sensory physiology.